The importance of the development of antigen detecting methods which enable prompt and simple measurement of a subject of analysis (e.g., antigen) among complicated samples and provide high reliability on measurement values is growing bigger and bigger. For example, the point of clinical diagnosis in the hospital emergency is that an unskilled technician can promptly and accurately check a patient's condition by performing complicated chemical or immunochemical analyses. These analyses are generally conducted by hospital staff or nurses who are not trained as a clinical chemist. Current clinical diagnosis systems in which blood samples are transferred to the hospital laboratory and then analyzed are not suitable when prompt laboratory findings are requested. Therefore, the measurement methods, in which analysis results can be provided in a short time and staff and equipment for conducting analyses are suitable for the request in the hospital diagnosis system, are required.
As the existing antigen detecting method, immunoassay is mainly used. Particularly, the immunoassay is generally used in detecting items of tumor markers. The immunoassay uses an antigen-antibody reaction, and can detect a desired material by using an antibody selectively binding to a material to be detected. The kinds and principles of representative immunoassays are as follows. Although the using methods and specific reaction conditions may be slightly different according to organs, basic principles thereof are almost the same as each other.
Particle immunoassay is a test that uses agglutination occurring by the binding of antigen and antibody. In most cases, the antigen or antibody is bound to latex, gelatin or the like, and this particle reacts to show agglutination, which is then measured. The measurement of agglutination may be made by measuring the absorption degree of light through turbidimetry or the scattering degree of light through nephelometry.
Enzyme immunoassay (EIA) is a test that uses an enzyme reaction to measure the binding of antigen and antibody. In most cases, an enzyme is previously bound to an antibody which binds to a material to be measured, to cause an antigen-antibody reaction. After that, a substrate is put into the bound enzyme to cause an enzyme reaction. Examples of a frequently used enzyme are alkaline phosphatase, horseradish peroxidase, β-galactosidase, etc. The products from the enzyme reaction are mostly materials having colors, which are measured by a spectrophotometer.
Radioimmunoassay is a test that uses radioisotopes to measure the binding of antigen and antibody. Radioisotopes are materials that are physically unstable, resulting in natural radioactive decay, and thus changed into stable materials, and produce radiation in this process. The radioisotopes are bound to a material, like a material to be measured, or an antibody reacting with the material to be measured, causing an antigen-antibody reaction. After the reaction is ended, the amount of radiation released from the reaction material is measured, thereby calculating the concentration of a desired material. The radioisotopes that are mostly used are 125I, 131I, 3H, 14C, 32P, etc. The radioimmunoassay had mostly been used in the past, but the use thereof has been decreasing since there is a risk of using a radioactive material and chemiluminescence immunoassay and the like have been developed.
Fluorescence immunoassay is a test that uses a fluorescence reaction to measure the binding of antigen and antibody. The fluorescence reaction refers to a reaction in which a molecule of a fluorescent material is excited when the fluorescent material absorbs a particular wavelength of light, and releases light having a different wavelength from the absorbed light when recovered to its original state. In the case where the fluorescence reaction is used for immunoassay, the fluorescent material is bound to a material, like a material to be measured, or an antibody reacting with the material to be measured, causing an antigen-antibody reaction. After the antigen-antibody reaction, the projection of light that has such a wavelength to cause a fluorescence reaction leads to the fluorescence in proportion to the amount of fluorescent material, and the amount of fluorescent material is used to calculate the concentration of the material to be measured.
Chemiluminescence immunoassay is a test that uses chemiluminescence to measure the binding of antigen and antibody. Chemiluminescence is the emission of light while the excited chemiluminescent material returns to the ground state, and is different from fluorescence in that the energy used to excite a molecule is not light but a chemical reaction. In the case where the chemiluminescence is used for immunoassay, the chemiluminescent material is bound to a material, like a material to be measured, or an antibody reacting with the material to be measured, causing an antigen-antibody reaction, in the same manner as other methods. After the reaction, a necessary chemical reaction is caused, and then the degree of luminescence emitted is measured, thereby calculating the concentration of material to be measured therefrom. Representative examples of the luminescent material are luminol, isoluminol, acridinium ester, etc.
The antigen detecting method which is to be used herein is based on Fluorescence immunoassay, and has been developed in order to overcome a problem of the conventional Fluorescence immunoassay in that reproducibility and reliability of data are poor since the measurement value of antigens is changed depending on the concentration of the sample and the reaction temperature. Accordingly, the present inventors have attempted to establish an antigen detection system which allows on-site diagnosis and is not interrupted by the concentration of a sample to be analyzed and the reaction temperature, based on Fluorescence immunoassay using an antigen-antibody reaction.
Throughout the entire specification, many papers and patent documents are referenced and their citations are represented. The disclosures of cited papers and patent documents are entirely incorporated by reference into the present specification, and the level of the technical field within which the present invention falls and details of the present invention are explained more clearly.